This invention is in the fields of neurology and pharmacology, and relates to drugs that can minimize brain injury due to various causes, such as traumatic head injury or crises such as stroke, cardiac arrest, or asphyxiation.
The compounds disclosed herein (referred to as "fluorenone" drugs) are all within a class of compounds that were first discovered, and recognized to be potentially useful for reducing brain damage, in the 1970's. A great deal of time, effort, and expense were devoted to these drugs, and they were extensively patented and studied by one of the world's largest pharmaceutical companies, Merck & Company, Inc.
However, these drugs were never commercialized in any way, at any time, by Merck or any other company. The primary discoverer and inventor, Dr. Edward Cragoe, Jr., retired from Merck years ago, without ever getting to see any of these drugs provide the much-needed public service of offering a treatment to reduce or prevent brain injury. The patents on these fluorenone drugs have either expired, or were deliberately allowed to lapse due to intentional non-payment of the maintenance fees. The expired or lapsed patents in this field which cover fluorenone compounds include U.S. Pat. Nos. 4,316,043 (issued in February 1982); 4,317,922 (March 1982); 4,337,354 (June 1982); 4,356,313 and 4,356,314 (both in October 1982); 4,604,396 (August 1986); 4,675,341 (June 1987); 4,731,471 and 4,731,472 (both in March 1988); 4,782,073 (November 1988); 4,797,391 (January 1989); and 4,835,313 (May 1989).
Other lapsed and abandoned US patents which disclose and claim methods of synthesizing such compounds include U.S. Pat. Nos. 4,605,760 and 4,605,761 (both issued in August 1986).
Still other lapsed and abandoned US patents which disclose fluorenone-type compounds that are not as closely related to the subject matter of this invention include U.S. Pat. Nos. 4,731,470 (March 1988); 4,769,370 (September 1988); and 4,777,281 (October 1988).
Not a single one of the "fluorenone" compounds covered in any of the patents listed above has ever been commercialized or made available to the public. As mentioned above, all of the patents listed above were allowed to lapse and expire, due to nonpayment of their maintenance fees.
Fluorenone Compounds
The compounds disclosed in the most relevant prior art belong to a class of compounds that are analogs (mostly in the form of ether or ester analogs) of R-(+)-(5,6-dichloro-2,3,9,9a-tetrahydro-7-hydroxy-9a-hydrocarbyl-1H-fluore n-3-one compounds. Their general chemical structure is: ##STR2##
where R, X, and Y.sup.1 and Y.sup.2 are various organic moieties as specified in the prior art patents.
These compounds are sometimes called "fluorene" compounds or derivatives, because they are contain a tri-cyclic structure called fluorene, which is shown in the Merck Index and in various articles cited therein which date back to the 1920's. The illustration of fluorene analog L-644,711, shown at the top of FIG. 1 herein, shows the conventional numbering used for the carbon atoms in the three-ring structure of fluorene.
It should be noted that fluorene has no relation to fluorine (the halogen atom), even though both words are pronounced the same. To avoid confusion, a fluorene compound which bears a double-bonded oxygen attached to one of the three ring structures can be called a "fluorenone" compound. All of the drugs discussed herein are fluorenone compounds, since they bear a double-bonded oxygen attached to the 3-carbon atom, as shown in FIG. 1.
Cragoe et al 1986 and Cragoe 1987, which are review articles, provide additional information on prior art fluorenone compounds. Briefly, Cragoe and his coworkers initially began working with compounds known as "indanones", which were demonstrated to have diuretic activity (i.e., they caused the excretion of large quantities of body fluids, via urine); see Woltersdorf et al 1977 and deSolms et al 1978. By systemically reducing body fluids via increased urine output, some of these drugs were shown to help reduce edema inside the brain after a traumatic brain injury. This reduction of edema inside the brain helped restore blood flow in an injured brain, as discussed in Cragoe et al 1986.
It was subsequently discovered and shown that some indanone compounds (which are bi-cyclic) could be further cyclized to generate tri-cyclic fluorenone compounds, which had increased activity in reducing brain edema without the unwanted systemic side effects produced on body tissues by diuretic agents; this was discussed in Cragoe 1987.
Accordingly, subsequent research by the Merck scientists focused on the tri-cyclic fluorenone compounds as potential neuroprotective drugs. That research in the late 1980's eventually settled on the L-644,711 compound as Merck's lead compound for in vitro and in vivo testing, since it was one of the most desirably active, effective, and selective compounds known at that time. In that particular compound, the "X" group attached to the 9a-position is a propyl group in the R(+) orientation, and the "R" group attached to the 7-carbon atom is a carboxymethyl group (HOOCCH.sub.2 --). The complete chemical name of compound L-644,711 is R(+)-[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluoren-7-yl)ox y]acetic acid, and it is shown as a starting reagent at the top of FIG. 1. It is also shown as compound (+)-5c in Cragoe et al 1986 (which summarizes the steps used to synthesize the entire series of fluorenone compounds), and as compound B-3(+) in Cragoe 1987 (which discusses the biological activities of various fluorenone compounds).
Samples of L-644,711 were provided by the Merck company to various researchers at medical schools and elsewhere, who tested it and reported on its potential for preventing brain damage (see, e.g., Kimelberg et al 1987 and 1989; Barron et al 1988; Trachtman et al 1989; Bednar et al 1992; and Kohut et al 1992).
However, as noted above, that line of research was abandoned within a few years. It did not lead to any commercialized compounds, and all of the US patents listed above were allowed to lapse and expire, due to non-payment of their maintenance fees.
L-644,711 was used as a "benchmark" compound in the new research disclosed herein. This new research, which was sponsored and funded by Cypros Pharmaceutical Corporation (Carlsbad, Calif.), identified a number of compounds that are markedly better than L-644,711 as a neuroprotective drug, as measured by appropriate biological assays.
Accordingly, the new compounds disclosed herein should be regarded as highly improved fluorenone compounds which perform markedly better than any of the prior art compounds disclosed in any of the patents cited above, or in any other publications that are known to the Inventors herein.
Background on Glial Cells and Traumatic Brain Edema
Inside the mammalian central nervous system (CNS, which includes the brain and spinal cord), cells are divided into two major categories: neurons, and glial cells. Neurons are cells which actually receive and transmit nerve signals. By contrast, the term "glial cells" includes a variety of supporting cells which help nourish and protect neurons, but which do not and cannot receive and transmit nerve signals. Glial cells are subdivided into various cell types, including: (1) astrocytes, which have cell shapes that resemble a star in certain respects, with a main central portion having various arms projecting outwardly from the central portion; (2) oligodendrocytes, which have several long projecting "dendrites", which usually wrap around certain portions of the neurons, to provide myelin sheaths which surround neuronal dendrites and axons; and (3) microglial cells, which are migratory cells that are part of the immune system inside the brain, and which collect and break down waste products, dead cells, and bacterial cells and viruses inside the brain tissue.
More information on glial cells, and on the interactions between glial cells and neurons inside the central nervous system, is contained in numerous reference books on neurology, such as Principles of Neural Science, 3rd edition, by E. Kandel & J. Schwartz (Elsevier Publishing, New York, 1991), a one-volume textbook, or Encyclopedia of Neuroscience, edited by G. Adelman (Birkhauser Publishing, Boston, 1987), a multi-volume treatise.
For convenience, most of the remaining discussion focuses on the brain, and on injuries or other insults to the head. However, it should be understood that astrocytes and other glial cells also exist and function in essentially the same manner in a mammalian spinal cord. Accordingly, the drugs disclosed herein are believed to be useful for reducing neuronal damage to a spinal cord as well as to a brain, as further discussed below.
The drugs discussed herein may have various effects on any type of glial cells. Astrocyte cells were selected and used for various tests disclosed herein for a number of reasons, as follows.
Astrocyte cells often swell after a head injury, and this cellular swelling can severely aggravate and multiply the extent and severity of brain damage resulting from an injury. The complete set of causes and aggravating factors which lead or contribute to astrocyte swelling and edema are not totally understood; however, a sequence of three important cellular reactions are known to be major contributing factors.
In the first step in this series of reactions, chloride ions (Cl.sup.-) begin entering astrocyte cells in abnormally large quantities by an active process, from surrounding extra-cellular fluids. These ions enter the cells through specialized chloride channels that pass through the astrocyte cell membranes.
In the second step, positively charged sodium ions (Na.sup.+) also begin entering the astrocyte cells in abnormally large quantities by a passive process, due to effect of the excess of negatively charged Cl.sup.- ions inside the cells.
In the third step, after the influx of ions into astrocyte cells creates an osmotic imbalance between the intracellular and extracellular fluids, water molecules begin seeping into the astrocyte cells, in an effort to re-establish proper osmotic balances across the cell membranes.
As a result of these processes (and possibly other contributing factors as well), the affected astrocyte cells become swollen due to the presence of large quantities of excess water. The medical term for this condition is "edema", which refers to swelling of cells or tissue caused by a combination of (a) entry of too much fluid into the cells or tissue, combined with (b) an inability of the cells or tissue to excrete or otherwise properly manage the excess fluid.
When astrocyte cells become swollen, they begin pressing against the capillaries that provide blood to the brain tissue. Capillary walls inside the brain are very thin and pliable; this is necessary to allow adequate quantities of glucose, oxygen, and other nutrients to permeate out from the blood and through the capillary walls, to provide nourishment to nearby neurons and glial cells.
Because of their thinness, capillary walls cannot significantly resist the pressure that is generated when astrocyte cells become edematous. Accordingly, astrocyte edema can severely restrict subsequent blood flow through capillaries that serve an affected region inside the brain. This reduction of capillary blood flow through the brain can quickly become catastrophic, and will lead to severe and possibly lethal brain damage, unless it is relieved quickly.
Astrocyte cells can also severely aggravate brain damage after a head injury, due to a second major factor. This factor arises from the fact that in a healthy brain, astrocyte cells help to "mop up" excess quantities of certain types of excitatory neurotransmitters, especially glutamate and aspartate. In a healthy brain, glutamate and/or aspartate are released by a neuron in order to transmit a nerve impulse to an adjacent neurons. After being released into a synaptic junction, a glutamate or aspartate molecule briefly binds to a receptor protein on the surface of the signal-receiving neuron. This interaction between the glutamate or aspartate transmitter molecule and the neuronal receptor provokes a cellular response, which causes ion channels in the signal-receiving neuron to briefly open and allow certain types of ions to enter the neuron. This influx of ions changes the chemical state of the neuron, thereby activating ("depolarizing") the neuron, and causing it to release its own set of neurotransmitter molecules at synapses with other neurons.
As soon as a neuron has been activated (i.e., depolarized), it activates its ion pumps and begins pumping ions back out of the cell, in order to regain its polarized state so it will be ready to receive another nerve impulse. This effort to regain a polarized "ready-to-fire" state requires a neuron to expend substantial amounts of energy. In effect, the "resting state" of a neuron is on a high-energy plateau; it can reach a "ready-to-fire" resting state only by pumping out large quantities of ions.
When glutamate or aspartate are used to transmit a nerve impulse, the glutamate or aspartate molecules quickly disengage from the receptor proteins and enter the synaptic fluid again. In a healthy brain, the large majority of glutamate and aspartate molecules which have been released from neuronal receptors in this manner are quickly pumped back inside the neurons that released those transmitter molecules, by a cellular transport system which requires energy to run. However, some glutamate and aspartate molecules are not handled properly by this neuronal pumping system, and they diffuse out of the synaptic junctions, in a manner comparable to a slowly dripping faucet. These errant neurotransmitters would pose a serious risk of creating unwanted and possibly destructive nerve impulses, if they were not promptly managed by other mechanisms.
To prevent uncontrolled nerve signals from being triggered by glutamate and aspartate molecules which have gradually leaked out of the synaptic junctions between neurons, astrocyte cells have developed a highly useful "mopping up" function. In simple terms, astrocyte cells will grab any glutamate or aspartate molecules they encounter, and pump those molecules into their cell interiors. Because astrocyte cells do not quickly metabolize and degrade these neurotransmitter molecules, the astrocyte cells gradually accumulate fairly large quantities of glutamate and aspartate molecules.
In a healthy brain, this is good and proper; the glutamate and aspartate molecules which are stored inside astrocyte cells do not harm those cells in any way. However, if a brain injury occurs which is severe enough to cause badly-stressed astrocytes to swell and suffer from edema, the stressed astrocytes can begin releasing their stored-up quantities of glutamate and aspartate. If this occurs, the newly-released glutamate and aspartate will begin contacting neurons again, triggering unwanted nerve impulses in uncontrolled ways and at the worst possible time. The neurons will already be under severe stress due to the brain injury which triggered the crisis, and as mentioned above, each time a neuron undergoes a depolarizing event, it immediately begins expending large quantities of energy in an effort to pump out the ions that entered it when the neuron "fired", so it can get ready to receive the next nerve impulse.
Accordingly, if a traumatic brain injury causes astrocyte cells to swell and begin releasing glutamate and aspartate into extracellular fluids inside the brain, matters can quickly go from bad to worse. An "excitotoxic cascade" of cell damage and death inside the brain can break free of the restraining limits which the brain normally uses to prevent over-excitation. These processes can severely aggravate brain damage, and often lead to the death of the victim.
These processes, and the correlations between cellular swelling and the release of glutamate and aspartate inside CNS tissue, are discussed in articles such as Bourke et al 1983 and Kimelberg et al 1990.
The terms "excitotoxic" and "excitotoxin" are used by neurologists to indicate that excitatory neurotransmitters, which play an essential role in a healthy brain, can become deadly neurotoxins in a brain suffering from a crisis. During and after an ischemic, hypoxic, or similar crisis, glutamate and aspartate both become excitotoxins, and can kill affected neurons through toxic over-excitation.
Accordingly, this invention discloses new compounds which are more potent and effective than any previously known compounds in reducing the release of excitotoxic quantities of glutamate and aspartate by glial cells (including astrocyte cells) following a CNS crisis.
Since these new compounds exert this effect, they are referred to herein as "GERI" compounds, where GERI is the acronym for "Glial Excitotoxin Release Inhibitors". This activity has been shown using an assay involving the release of radiolabelled aspartate by osmotically-stressed astrocytoma cells, described in detail in Example 23, below.
Referring to these compounds as "Glial Excitotoxin Release Inhibitors" does not imply that their GERI function is their only known useful activity. A correlation was observed during the astrocyte assays, indicating that the potency of various fluorenone analogs in inhibiting excitotoxin release by stressed astrocyte cells apparently correlates with their ability to also reduce edematous swelling by the cells. This observation suggests that (i) the GERI class of fluorenone analogs may be extremely useful in preventing or reducing CNS damage caused by various types of crises as discussed below; and, (ii) the D-aspartate release assay may be useful as an easily measured, readily quantifiable indicator of a GERI compound's ability to minimize astrocyte swelling, and possibly relieve and reduce elevations in intracranial pressure as well, following a head trauma or other CNS crisis. If desired, such correlations can be further elucidated by quantitative measurements of edema in astrocytes, using in vitro assays such as described in O'Connor et al 1993.
Based on the assays done to date, which include in vivo animal tests as disclosed in Examples 24 and 25, the GERI compounds disclosed herein are believed to be effective and potent neuroprotective compounds, which can be used to reduce and prevent damage to a mammalian brain and/or spinal cord due to any of the following causes and etiologies:
1. physical trauma to the head or spinal cord, as can occur in automobile accidents, bad falls, sports injuries, etc.; PA1 2. a brain concussion, which can occur due to physical trauma to the head, and in certain other types of situations involving rapid acceleration or deceleration of the head; PA1 3. stroke, including ischemic stroke caused by thrombosis or embolism, regardless of where a blood clot or other embolus originates in the body; PA1 4. other disruptions of proper blood flow through the brain, such as (i) cerebral hemorrhage; (ii) general circulatory failure or disruption, such as caused by cardiac arrest; (iii) hemodynamic shock, such as caused by loss of blood due to injury or hemorrhage elsewhere in the body; (iv) vasculatory damage, as can be caused by vascular disease, certain types of bacterial, viral, or other microbial infection, and other comparable causes; (v) cerebral or spinal tumors; and, (vi) glial cell swelling caused by infections (such as viral, bacterial, or other microbial meningitis, encephalitis, or encephalomyelitis, Reyes syndrome, or AIDS) or other mechanisms, such as hydrocephalus; PA1 5. hypoxic injury to the brain (i.e., inadequate oxygen supply), which arises as a direct result of any ischemic crisis, and which can also be caused by respiratory disruption, as occurs during incipient drowning or suffocation, carbon monoxide poisoning, etc.; and, PA1 6. post-operative brain injury or stress, as can be caused by neurosurgery, or by cardiopulmonary bypass for a prolonged period.
Accordingly, one object of this invention is to disclose new drugs which can reduce and minimize brain and spinal damage following traumatic injuries.
Another object of this invention is to disclose new drugs which can reduce and minimize brain damage following various types of medical crises, such as strokes, cardiac arrest, and infective or inflammatory processes such as meningitis, encephalitis, or encephalomyelitis.
Another object of this invention is to disclose a class of drugs which can minimize astrocyte swelling inside the brain, following an injury or infection that affects the head or central nervous system.
Another object of this invention is to disclose a class of drugs which can be used to minimize the release, by astrocyte cells, of excitatory neurotransmitters (especially glutamate and aspartate) inside the brain or spinal cord after a head or spinal injury.
In addition, another object of this invention is to disclose certain drugs that can markedly outperform and improve upon L-644,711, a preferred compound from the "fluorenone" class of drugs which was extensively studied but never commercialized or made available to the public. Thus, the compounds of this invention possess greater potency for the treatment of brain and spinal cord injury, and they also enjoy a broad scope of biochemical mechanism of action.
These and other objects of the invention will become more apparent through the following summary, drawings, and description of the preferred embodiments.